The fixation of molecular nitrogen from the air, i.e. its reduction to yield ammonia and other biologically useful forms of nitrogen is an extremely important process in the biosphere. However, it can be carried out by only a limited number of organisms. The majority of organisms must obtain nitrogen in a combined form such as nitrate, ammonia or amino acids. Most leguminous plants e.g. clover and alfalfa can fix atmospheric nitrogen. but the process requires the symbiotic action of the host plant and bacteria present in the root nodules of the plant. The bacteria involved in nitrogen fixation belong mainly to the genus Rhizobium, and these bacteria enter the cortical parenchyma of the root where they give rise to nodules. The nodules are highly organized structures with membraneous sacks containing colonies of bacteria. Supplied with carbon compounds from photosynthesis by the plant, the bacteria fix nitrogen, producing ammonia which is provided to the plant for amino acid biosynthesis, thus aiding protein formation in the plant. Leguminous plants can therefore be grown without synthetic nitrogen fertilizers which represents a considerable financial saving for the farmer since it allows for protein production on a cost effective basis.
The process of nitrogen fixation is the basis for the use of clover and alfalfa in crop rotation, since the growth of such plants increases the utilizable nitrogen in the soil.
Legume seeds which have been coated with Rhzobium species are currently available commercially. This allows for the formation of nitrogen fixing root nodules to be formed as soon as the seeds germinate. The seeds are coated with naturally occurring strains which have been isolated from soil and selected for their efficiency in fixing nitrogen and their ability to compete with other bacterial strains in the soil.
Evidence indicates that the nitrogen-fixing capacity of the Rhizobiumlegume symbosis is influenced by the amount of photosynthate available to the bacteria in the root nodule (Hardy and Havelka, (1975) In: Nutman P.S. (ed) International biological programme : symbotic nitrogen fixation in plants. Cambridge University Press, New York, pp 421-439; Havelka and Hardy (1976) In : Newton W.E., Nyman. C.Y. (eds) Proceedings of the first international symposium on nitrogen fixation. Washington State University Press, Pullman, Washington. pp 456-475; and Ryle et al (1979) J. Exp. Bot. 30 : 135-144). It is widely thought that one of the key limiting steps in symbiotic nitrogen-fixation is the availability of the carbon (i.e. energy) source made available to the bacteria.
The inventors of the present invention do not agree fully with this hypothesis but believe that the limiting factor may not be the availability of a carbon source but rather the transport of the carbon source into the bacteria in the root nodule.
Tricarboxylic acid (TCA) cycle intermediates have been implicated as the major carbon source supplied to bacteroids (i.e. the differentiated form of bacteria which fixes nitrogen in the root nodule) in pea and clover symbiosis. The evidence for this is based on the fact that dicarboxylic acid transport mutants of Rhizobium leguminosarum (Finan et al (1983) J. Bacteriol 148, 193-202; Glenn and Brewin (1981) J. Gen. Microbiol. 126, 237-242) and R. Trifolii (Ronson et al (1981) PNAS (USA) 78, 4284-4288) form ineffective nodules despite being able to utilize other carbon sources. In R. meliloti, a functional TCA cycle is necessary to support nitrogen-fixation in the bacteroid as shown by the fact that mutants defective in succinate dehydrogenase (Gardiol et al (1982) J. Bacteriol. 151 1621-1623) and alpha-ketoglutarate dehydrogenase (Duncan and Fraenkel (1979) J. Bacteriol. 37 415-419) form ineffective nodules.
Dicarboxylic acid transport genes from R. leguminosarum have been isolated (Ronson et. al. (1984) J. Bacteriol. 160 903-909) but they did not lead to a significant effect on the symbiotic response of the wild-type strain.
The strain formerly known as Rhizobium japonicum has been officially re-classified as Bradyrhizobium japonicum and references to that strain in this specification have been amended accordingly. The term "Rhizobium species" as used herein includes both Rhizobium and bradyrhizobium species.